Angiopoietin-2 (Ang2) is a 70 kDa secreted ligand whose increased expression has been implicated in a range of diseases, including cancer, sepsis and adult respiratory distress syndrome (1, 2). The primary receptor for Ang2 is the transmembrane tyrosine kinase Tie2 (3) that is expressed mainly on vascular endothelial cells and myeloid cells (1, 4). Ang2 plays an important role in vascular remodeling during development but in adult tissues Ang2 concentrations are usually low. An increase in Ang2 levels in disease allows the molecule to compete for binding to a common interface on Tie2 with the related agonist Ang1 (3). Ang1 is a protective protein constitutively produced by perivascular cells which maintains blood vessel function and quiescence by suppressing inflammation, vessel leakage and endothelial apoptosis (1, 5). Antagonism of Ang1 by Ang2 blocks the pro-quiescent effects of Ang1 and contributes to Ang2-induced vessel remodelling, inflammation, leakage and oedema. In addition to its actions on endothelial Tie2, Ang2 has a number of other effects relevant to disease. For example, the ligand has recently been shown to bind and activate endothelial integrins to promote sprouting angiogenesis (6), and Ang2 acts on tumour infiltrating Tie2-expressing monocytes to promote tumourigenesis (7, 8).
Because of its involvement in multiple disease processes there have been considerable efforts to develop inhibitors of Ang2, including antibodies and aptamers (9-11). Results from studies with these and related molecules have been encouraging, with reports of Ang2 inhibitors promoting tumor regression and suppressing of metastatic disease in cancer, and decreasing leukocyte infiltration and vascular remodeling in airway inflammation (7, 10, 12, 13).
A complementary approach to the use of antibodies for blocking pathological levels of ligands is the cytokine or ligand trap (14). These molecules are formed from receptor ectodomain fragments, usually administered as soluble fusion proteins, which sequester the target ligand. Examples of ligand traps in clinical use include Etanercept, a soluble form of tumour necrosis factor-α receptor and Aflibercept, a chimeric fusion protein of fragments of vascular endothelial growth factor receptor-1 and -2 (15). There are significant advantages to ligand traps. Usually they are smaller and have better tissue penetration than antibodies, they already recognize the biologically active part of the target and generally do not require protection from the immune system. A ligand trap specific for Ang2 would be an attractive therapeutic. However the natural receptor for Ang2, Tie2, binds to the protective ligand Ang1 equally well or even better than it does to Ang2 (3, 16, 17).
One of the most effective strategies for engineering new protein functionality is directed protein evolution (18, 19). This process essentially recapitulates the selection and accumulation of desirable mutations that occurs in natural evolution over millions of years, but over a period of weeks in the laboratory. Directed evolution involves repeated rounds of library construction, usually in vitro, expression of the mutant forms of the target protein and selection. Unfortunately this iterative approach to in vitro generation and searching of sequence space is frequently both difficult and labour intensive. B cell lines that constitutively diversify their immunoglobulin variable (IgV) regions by somatic hypermutation (SHM) (20) allow for coupling of diversification and selection of novel antibody specificities. The genetic variation within the Ig genes, introduced by the action of activation induced deaminase (AID) is coupled to the selectable expression of surface Ig on individual cells (21). More recently such cell lines have been used to evolve variants of exogenously expressed green fluorescent protein (22, 23). However, in theory this strategy has enormous potential for directed evolution of a wide range of proteins if the desired phenotype can be selected for in B lines.
There is thus still a need for an improved inhibitor of Ang2. In particular, there is a need for a polypeptide angiopoietin inhibitor which is capable of discriminating between Ang2 and Ang1.